1. Field of the Invention
The present invention relates generally to the fields of analytical chemistry and biochemistry. More specifically, the present invention relates to using matrices comprising viscous liquids and light absorbing particles for analysis of microorganisms by laser desorption mass spectrometry.
2. Description of the Related Art
In the last decade, matrix-assisted laser desorption/ionization (MALDI) has become one of the most important ionization method used for biological mass spectrometry..sup.1-3 Before the invention of matrix-assisted laser desorption/ionization, several desorption techniques had been used to produce ions. These include field desorption,.sup.4 plasma desorption,.sup.5 laser desorption,.sup.6 SIMS.sup.7 and fast atom bombardment (FAB)..sup.8 Most important is the introduction of the liquid matrix in FAB, opening up a new era of biochemical applications of mass spectrometry.
The requirements for a matrix-assisted laser desorption/ionization matrix include absorption of the laser light, promotion of ionization and dispersion of the energy deposited on the sample in order to produce intact analyte ions. A useful matrix would also be compatible with a broad range of laser wavelengths. Further, matrix-assisted laser desorption/ionization experiments are thought to require co-crystallization of analytes with solid matrices such as 2,5-dihydroxybenzoic acid and 4-hydroxy-.alpha.-cyanocinnamic acid. There are several problems associated with these crystallization processes. The tendency to form inhomogeneous crystalline phases leads to the existence of "sweet spots". The ion signal also decreases with the number of laser shots impinging on a given sample spot. In contrast, no crystallization process is involved in liquid matrix methods. Analytes are dissolved in the liquid and are evenly distributed. This provides a homogeneous response and extends the sample lifetime at a given spot as the matrix backfills the cavities and dilutes molecules damaged by the laser beam.
Several approaches toward the use of liquids have been made. One approach is the use of laser light-absorbing liquids as matrices. For example, 3-nitrobenzyl alcohol has been evaluated with a 266 nm laser or a N.sub.2 laser at 337 nm..sup.9-11 Glycerol whose O--H stretching mode has strong absorption in the 3 .mu.m region, has been used with IR lasers at 2.94 .mu.m and 10.6 .mu.m to provide mass spectra of lysozyme at 14,300 Da..sup.12-14 Alternatively, for liquid matrices that do not absorb the laser wavelength, strongly absorbing compounds such as rhodamine 6G (absorbing visible laser light at 532 nm).sup.15,16 and 2-cyano-5-phenyl-2,4-pentadienoic acid (absorbing UV laser light).sup.17 has been dissolved in the matrix.
Another similar approach to coupling laser irradiation into liquid matrices is to suspend small particles in the liquids. Tanaka et al. were the first group to report UV-matrix-assisted laser desorption/ionization mass spectra of proteins as large as chymotrypsinogen (.about.25,700 Da) by suspending 30 nm cobalt particles in glycerol..sup.18 Recently, Hillenkamp et al. have evaluated various combinations of particles (TiN, Pb, graphite) and solvents (glycerol, 3-nitrobenzylalcohol, triethanolamine, DMSO) to obtain mass spectra of peptides and proteins using a 337 nm and 1064 nm lasers..sup.19 Sunner et al..sup.20 and Zenobi et al..sup.21,22 reported mixing graphite particles with glycerol and diethanolamine. Liquid matrices consisting of glycerol and K.sub.4 Fe(CN).sub.6 ! or Na.sub.4 Fe(CN).sub.6 ! have been investigated by Allmaier et al..sup.23,24
The rapid and accurate characterization of microorganisms is important in many applications including food production, disease diagnoses and biohazard recognition. Fast atom bombardment, which is well known for its use of a liquid matrix has been used worldwide to differentiate bacteria based on profiles of cellular polar lipids..sup.25-27 Furthermore, spectra of polar lipids may be obtained directly from lysed cells without extraction or processing. The liquid matrices serve a role to separate soluble compounds in microorganisms and to bring the sample to the surface. Using liquid matrices for matrix-assisted laser desorption/ionization, mass spectra of peptides have been obtained that are similar to those obtained by fast atom bombardment..sup.20
The prior art is deficient in the lack of application of particle/liquid matrices to improve the use of matrix-assisted laser desorption ionization mass spectrometry and for the detection of different phospholipids and other biomarkers directly from microorganisms on different kinds of mass spectrometers. The present invention fulfills this long-standing need and desire in the art.